mTOR ligands and polynucleotides encoding mTOR ligands

ABSTRACT

The invention relates to kinase ligands and polyligands. In particular, the invention relates to ligands, homopolyligands, and heteropolyligands that modulate mTOR activity. The ligands and polyligands are utilized as research tools or as therapeutics. The invention includes linkage of the ligands and polyligands to a cellular localization signal, epitope tag and/or a reporter. The invention also includes polynucleotides encoding the ligands and polyligands.

This application claims benefit of priority to U.S. provisionalapplication 60/868,539, filed Dec. 4, 2006, the contents of which areincorporated by reference herein.

FIELD OF INVENTION

The invention relates to mammalian kinase ligands, substrates andmodulators. In particular, the invention relates to polypeptides,polypeptide compositions and polynucleotides that encode polypeptidesthat are ligands, substrates, and/or modulators of mTOR. The inventionalso relates to polyligands that are homopolyligands orheteropolyligands that modulate mTOR activity. The invention alsorelates to ligands and polyligands tethered to a subcellular location.

This application has subject matter related to application Ser. No.10/724,532 (now U.S. Pat. No. 7,071,295), Ser. No. 10/682,764(US2004/0185556, PCT/US2004/013517, WO2005/040336), Ser. No. 11/233,246,and US20040572011P (WO2005116231). Each of these patents andapplications is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Kinases are enzymes that catalyze the addition of phosphate to amolecule. The addition of phosphate by a kinase is calledphosphorylation. When the kinase substrate is a protein molecule, theamino acids commonly phosphorylated are serine, threonine and tyrosine.Phosphatases are enzymes that remove phosphate from a molecule. Theremoval of phosphate is called dephosphorylation. Kinases andphosphatases often represent competing forces within a cell to transmit,attenuate, or otherwise modulate cellular signals and cellular controlmechanisms. Kinases and phosphatases have both overlapping and uniquenatural substrates. Cellular signals and control mechanisms, asregulated by kinases, phosphatases, and their natural substrates are atarget of research tool design and drug design.

Rapamycin is a triene macrolide antibiotic, produced by Streptomyceshygroscopicus, and which demonstrates anti-fungal, anti-inflammatory,anti-tumor and immunosuppressive properties. Rapamycin also indirectlyinhibits the activity of the protein, mTOR, (mammalian target ofrapamycin) which, under abnormal conditions, can promote tumor growth.There are two rapamycin analogs, RAD001 and CCI-779, that have shownanticancer activity in clinical trials. It is also desirable to developdirect inhibitors of mTOR as potential therapeutics.

Mammalian target of rapamycin (mTOR), RAFT1, and FRAP are the sameenzyme, herein referred to as mTOR. mTOR can phosphorylate serine andthreonine residues in protein or peptide substrates. Some cellularsubstrates of mTOR have been identified and are referenced in Brunn etal. 1997 J Biol Chem 272:32547-50; Burnett et al. 1998 Proc Natl AcadSci USA 95:1432-7; Carlson et al. 2004 Biochem Biophys Res Commun316:533-9; Carraway et al. 2004 Breast Cancer Res. 6:219-224; Gringas etal. 1999 Genes & Dev 13:1422-37; Isotani et al. 1999 J Biol Chem274:34493-8; Minami et al. 2001 Genes to Cells 6:1003-15; Mothe-Satneyet al. 2000 J Biol Chem 275:33836-43; Peterson et al. 2000 J Biol Chem275:7416-23; Yokogami et al. 2000 Current Biology 10:47-50. Whileindividual substrates or ligands have been identified and studied, mixedligands linked together as polyligands that modulate mTOR activity havenot been demonstrated before this invention. An aspect of the inventionis to provide novel, modular, inhibitors of mTOR activity by modifyingone or more natural substrates by truncation and/or by amino acidsubstitution. A further aspect of the invention is the subcellularlocalization of an mTOR inhibitor, ligand, or polyligand by linking to asubcellular localization signal.

Design and synthesis of polypeptide ligands that modulatecalcium/calmodulin-dependent protein kinase and that localize to thecardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J BiolChem (2003) 278:25063-71). Ji et al. accomplished this by generatingexpression constructs that localized calcium/calmodulin-dependentprotein kinase inhibitory polypeptide ligands to the sarcoplasmicreticulum by fusing a sarcoplasmic reticulum localization signal derivedfrom phospholamban to a polypeptide ligand. See also U.S. Pat. No.7,071,295.

DETAILED DESCRIPTION OF POLYPEPTIDE AND POLYNUCLEOTIDE SEQUENCES

SEQ ID NOS:1-6 are example polyligands and polynucleotides encodingthem.

Specifically, the mTOR polyligand of SEQ ID NO:1 is encoded by SEQ IDNO:2 and SEQ ID NO:3, wherein the codons have been optimized formammalian expression. SEQ ID NO:3 includes flanking restriction sites.SEQ ID NO:1 is an embodiment of a polyligand of the structureA-S1-B-S2-C-S3-D, wherein A is SEQ ID NO:22, B is SEQ ID NO:54, C is SEQID NO:24, and D is SEQ ID NO:31, wherein Xaa is alanine, and wherein S1is a spacer of the amino acid sequence PAAA, and S2 is a spacer of aminoacid sequence EFPGGG, and S3 is a spacer of the amino acid sequencePAGA. A polyligand of structure A-S1-B-S2-C-S3-D is also called herein aheteropolyligand, shown generically in FIG. 4D.

SEQ ID NO:4 is an embodiment of a polyligand of the structureX-S4-Y-S5-Z-S6-E, wherein X is SEQ ID NO:23, Y is SEQ ID NO:16, Z is SEQID NO:15, and E is SEQ ID NO:14, wherein Xaa is alanine, and wherein S4is a spacer of amino acid sequence AAA, S5 is a spacer of the amino acidsequence GGGG, and S6 is a spacer of the amino acid sequence AAAA. ThemTOR polyligand of SEQ ID NO:4 is encoded by SEQ ID NO:5 and by SEQ IDNO:6, wherein the codons have been optimized for mammalian expression.SEQ ID NO:6 includes flanking restriction sites. A polyligand ofstructure X-S4-Y-S5-Z-S6-E is also called herein a heteropolyligand,shown generically in FIG. 4E.

SEQ ID NOS:7-13 are full length mTOR protein substrates. These sequenceshave the following public database accession numbers: NP003152,BAA34402, NP446309, NP644805, AAB27175, NP_(—)004086, and P42345. Eachof the sequences represented by these accession numbers is incorporatedby reference herein. In SEQ ID NOS:7-13, the positions of the aminoacid(s) phosphorylatable by mTOR are represented by Xaa. In wild-typeproteins, Xaa is serine or threonine. In the ligands of the invention,Xaa is any amino acid.

SEQ ID NOS:14-55 are peptide subsequences or partial sequences of SEQ IDNOS:7-13, which represent examples of kinase active site blocker peptideligand sequences where the location of the mTOR phosphorylatable serineor threonine in the natural polypeptide is designated as Xaa.

SEQ ID NOS:14-55 represent examples of monomeric polypeptide ligandsequences.

Amino acid sequences containing Xaa encompass polypeptides where Xaa isany amino acid.

DETAILED DESCRIPTION OF DRAWINGS

FIGS. 1A-1C show examples of homopolymeric ligands without spacers.

FIGS. 2A-2C show examples of homopolymeric ligands with spacers.

FIGS. 3A-3E show examples of heteropolymeric ligands without spacers.

FIGS. 4A-4F show examples of heteropolymeric ligands with spacers.

FIGS. 5A-5G show examples of ligands and polymeric ligands linked to anoptional epitope tag.

FIGS. 6A-6G show examples of ligands and polymeric ligands linked to anoptional reporter.

FIGS. 7A-7G show examples of ligands and polymeric ligands linked to anoptional localization signal.

FIGS. 8A-8G show examples of ligands and polymeric ligands linked to anoptional localization signal and an optional epitope tag.

FIGS. 9A-9G show examples of gene constructs where ligands andpolyligands are linked to an optional localization signal, an optionalepitope tag, and an optional reporter.

FIGS. 10A-10D show examples of vectors containing ligand geneconstructs.

FIG. 11 shows an example of a sequential cloning process useful forcombinatorial synthesis of polyligands.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to polypeptide ligands and polyligands for mTOR.Various embodiments of the mTOR ligands and polyligands are representedin SEQ ID NOS:1-55. More specifically, the invention relates to ligands,homopolyligands, and heteropolyligands that comprise any one or more ofSEQ ID NOS:14-55. Additionally, the invention relates to ligands andpolyligands comprising one or more subsequences (partial sequences) ofSEQ ID NOS:7-13 or any portion thereof. Furthermore, the inventionrelates to polyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%,98% and 99% sequence identity to a polyligand comprising one or more ofSEQ ID NOS:14-55 or any portion thereof. Furthermore, the inventionrelates to polyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%,98% and 99% sequence identity to a polyligand comprising one or morepartial sequences of SEQ ID NOS:7-13.

Polyligands, which can be homopolyligands or heteropolyligands, arechimeric ligands composed of two or more monomeric polypeptide ligands.An example of a monomeric ligand is the polypeptide represented by SEQID NO:19, wherein Xaa is any amino acid. SEQ ID NO:19 is a selectedsubsequence of wild-type full length SEQ ID NO:10, wherein the aminoacid corresponding to Xaa in the wild-type sequence is a serine orthreonine phosphorylatable by mTOR. An example of a homopolyligand is apolypeptide comprising a dimer or multimer of SEQ ID NO:19, wherein Xaais any amino acid. An example of a heteropolyligand is a polypeptidecomprising SEQ ID NO:14 and one or more of SEQ ID NOS:15-55, wherein Xaais any amino acid. There are numerous ways to combine SEQ ID NOS:14-55into homopolymeric or heteropolymeric ligands. Furthermore, there arenumerous ways to combine additional partial sequences of SEQ ID NOS:7-13with each other and with SEQ ID NOS:14-55 to make polymeric ligands.

The polyligands of the invention optionally comprise spacer amino acidsbefore, after, or between monomers. SEQ ID NO:1 is an embodiment of apolyligand of the structure A-S1-B-S2-C-S3-D, wherein A is SEQ ID NO:22,B is SEQ ID NO:54, C is SEQ ID NO:24, and D is SEQ ID NO:31, wherein Xaais alanine, and wherein S1, S2, and S3 are spacers. This inventionintends to capture all combinations of homopolyligands andheteropolyligands without limitation to the examples given above orbelow. In this description, use of the term “ligand(s)” encompassesmonomeric ligands, polymeric ligands, homopolymeric ligands and/orheteropolymeric ligands.

Monomeric ligands can be categorized into types. One type of monomericligand is a polypeptide where at least a portion of the polypeptide iscapable of being recognized by mTOR as a substrate or pseudosubstrate(active site blocker). The portion of the polypeptide capable ofrecognition is termed the recognition motif. In the present invention,recognition motifs can be natural or synthetic. Examples of recognitionmotifs are well known in the art and include, but are not limited to,naturally occurring mTOR substrates and pseudosubstrate motifs (SEQ IDNOS:14-55 and partial sequences of SEQ ID NOS:7-13 containing arecognition motif). Another type of monomeric ligand is a polypeptidewhere at least a portion of the polypeptide is capable of associatingwith mTOR at a substrate or pseudosubstrate docking site (docking siteblocker). A docking site type of monomeric ligand prevents mTORsubstrate phosphorylation by interfering with substrate association andalignment.

A polymeric ligand comprises two or more monomeric ligands linkedtogether.

A homopolymeric ligand is a polymeric ligand where each of the monomericligands is identical in amino acid sequence, except that aphosphorylatable residue may be substituted or modified in one or moreof the monomeric ligands.

A heteropolymeric ligand is a polymeric ligand where some of themonomeric ligands do not have an identical amino acid sequence.

The ligands of the invention are optionally linked to additionalmolecules or amino acids that provide an epitope tag, a reporter, and/ora cellular localization signal. The cellular localization signal targetsthe ligands to a region of a cell. The epitope tag and/or reporterand/or localization signal may be the same molecule. The epitope tagand/or reporter and/or localization signal may also be differentmolecules.

The invention also encompasses polynucleotides comprising a nucleotidesequence encoding ligands, homopolyligands, and heteropolyligands. Thenucleic acids of the invention are optionally linked to additionalnucleotide sequences encoding polypeptides with additional features,such as an epitope tag, a reporter, and/or a cellular localizationsignal. The polynucleotides are optionally flanked by nucleotidesequences comprising restriction endonuclease sites and othernucleotides needed for restriction endonuclese activity. The flankingsequences optionally provide unique cloning sites within a vector andoptionally provide directionality of subsequence cloning. Further, thenucleic acids of the invention are optionally incorporated into vectorpolynucleotides. The ligands, polyligands, and polynucleotides of thisinvention have utility as research tools and/or therapeutics.

Detailed Description of the Invention

The present invention relates to ligands and polyligands that are mTORmodulators. Various embodiments of ligands and polyligands arerepresented in SEQ ID NOS:1-55. Polyligands are chimeric ligandscomprising two or more monomeric polypeptide ligands. An example of amonomeric ligand is the polypeptide represented by SEQ ID NO:30, whereinXaa is any amino acid. SEQ ID NO:30 is a selected subsequence ofwild-type full length SEQ ID NO:7, wherein the amino acid correspondingto Xaa in the wild-type sequence is a serine or threoninephosphorylatable by mTOR. Another example of a monomeric ligand is thepolypeptide represented by SEQ ID NO:55. Another example of a monomericligand is the polypeptide represented by SEQ ID NO:46. Each of SEQ IDNOS:14-55 represents an individual polypeptide ligand in monomeric form,wherein Xaa is any amino acid. SEQ ID NOS:14-55 are selected examples ofsubsequences (partial sequences) of SEQ ID NOS:7-13, however, otherpartial sequences of SEQ ID NOS:7-13 containing a recognition motif mayalso be utilized as monomeric ligands. Monomeric ligand subsequences ofSEQ ID NOS:7-13 may be wild-type subsequences. Additionally, monomericligand subsequences of SEQ ID NOS:7-13 may have the mTORphosphorylatable amino acids replaced by other amino acids. Furthermore,monomeric ligands and polyligands may have at least about 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity to a ligand comprising anamino acid sequence in one or more of SEQ ID NOS:14-55. Furthermore,monomeric ligands and polyligands may have at least about 80%, 85%, 90%,95%, 96%, 97%, 98% and 99% sequence identity to a subsequence of SEQ IDNOS:7-13.

An example of a homopolyligand is a polypeptide comprising a dimer ormultimer of SEQ ID NO:24, wherein Xaa is any amino acid. Another exampleof a homopolyligand is a polypeptide comprising a dimer or multimer ofSEQ ID NO:17, wherein Xaa is any amino acid.

An example of a heteropolyligand is a polypeptide comprising SEQ IDNO:55 and one or more of SEQ ID NOS:14-54, wherein Xaa is any aminoacid. There are numerous ways to combine SEQ ID NOS:14-55 intohomopolymeric or heteropolymeric ligands. Furthermore, there arenumerous ways to combine additional partial sequences of SEQ ID NOS:7-13with each other and with SEQ ID NOS:14-55 to make polymeric ligands.

Polyligands may comprise any two or more of SEQ ID NOS:14-55, whereinXaa is any amino acid. SEQ ID NOS:14-55 are selected examples of partialsequences of SEQ ID NOS:7-13, however, additional partial sequences,wild-type or mutated, may be utilized to form polyligands. The instantinvention is directed to all possible combinations of homopolyligandsand heteropolyligands without limitation.

SEQ ID NOS:7-13 show proteins that contain at least one serine orthreonine residue phosphorylatable by mTOR, the positions of which arerepresented by Xaa. Since mTOR autophosphorylates, mTOR itself isincluded as a substrate. SEQ ID NOS:14-55 are partial sequences of SEQID NOS:7-13 where, again, the locations of the mTOR phosphorylatableresidues are represented by Xaa. In nature, Xaa is, generally speaking,serine or threonine. In one embodiment of the instant invention, Xaa canbe mutated to any amino acid. Ligands where Xaa is serine or threoninecan be used as part of a polyligand, however in one embodiment, at leastone phosphorylatable serine or threonine is replaced with or mutated toanother amino acid, such as one of the naturally occurring amino acidsincluding, alanine, aspartate, asparagine, cysteine, glutamate,glutamine, phenylalanine, glycine, histidine, isoleucine, leucine,lysine, methionine, proline, arginine, valine, tryptophan, or tyrosine.The Xaa may also be a non-naturally occurring amino acid. In anotherembodiment, the mTOR phosphorylatable serine(s) or threonine(s) arereplaced by alanine. The ligands and polyligands of the invention aredesigned to modulate the endogenous effects of mTOR.

In general, ligand monomers based on natural mTOR substrates are builtby isolating a putative mTOR phosphorylation recognition motif in a mTORsubstrate. Sometimes it is desirable to modify or mutate thephosphorylatable residue to an amino acid other than serine orthreonine. Additional monomers include the mTOR recognition motif aswell as amino acids adjacent and contiguous on either side of the mTORrecognition motif. Monomeric ligands may therefore be any lengthprovided the monomer includes the mTOR recognition motif. For example,the monomer may comprise an mTOR recognition motif and at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30-100 or more amino acids adjacent to therecognition motif.

For example, in one embodiment, the invention comprises an inhibitor ofmTOR comprising at least one copy of a peptide selected from the groupconsisting of:

-   a) a peptide at least 80% identical to a peptide comprising amino    acid residues corresponding to amino acid residues 406-415 of SEQ ID    NO:7, wherein the amino acid residue corresponding to amino acid    residue 412 of SEQ ID NO:7 is an amino acid residue other than    serine or threonine;-   b) a peptide at least 80% identical to a peptide comprising amino    acid residues corresponding to amino acid residues 405-418 of SEQ ID    NO:7, wherein the amino acid residue corresponding to amino acid    residue 412 of SEQ ID NO:7 is an amino acid residue other than    serine or threonine;-   c) a peptide at least 80% identical to a peptide comprising amino    acid residues corresponding to amino acid residues 402-423 of SEQ ID    NO:7, wherein the amino acid residue corresponding to amino acid    residue 412 of SEQ ID NO:7 is an amino acid residue other than    serine or threonine; and-   d) a peptide at least 80% identical to a peptide comprising amino    acid residues corresponding to amino acid residues 399-424 of SEQ ID    NO:7, wherein the amino acid residue corresponding to amino acid    residue 412 of SEQ ID NO:7 is an amino acid residue other than    serine or threonine.

In another embodiment, the invention encompasses an inhibitor of mTORselected from the group consisting of

-   a) a polypeptide comprising a partial sequence of SEQ ID NO:7,    wherein the partial sequence includes a mutation of at least one    amino acid residue at a position corresponding to amino acid residue    412.-   b) a polypeptide comprising a partial sequence of SEQ ID NO:8,    wherein the partial sequence includes a mutation of at least one    amino acid residue at a position corresponding to amino acid residue    401.-   c) a polypeptide comprising a partial sequence of SEQ ID NO:9,    wherein the partial sequence includes a mutation of at least one    amino acid residue at a position corresponding to amino acid residue    36, 45, 64, 69, and/or 82.-   d) a polypeptide comprising a partial sequence of SEQ ID NO:10,    wherein the partial sequence includes a mutation of at least one    amino acid residue at a position corresponding to amino acid residue    37 and/or 46.-   e) a polypeptide comprising a partial sequence of SEQ ID NO:11,    wherein the partial sequence includes a mutation of at least one    amino acid residue at a position corresponding to amino acid residue    727.-   f) a polypeptide comprising a partial sequence of SEQ ID NO:12,    wherein the partial sequence includes a mutation of at least one    amino acid residue at a position corresponding to amino acid residue    307.-   g) a polypeptide comprising a partial sequence of SEQ ID NO:13,    wherein the partial sequence includes a mutation of at least one    amino acid residue at a position corresponding to amino acid residue    2481.

As used herein, the terms “correspond(s) to” and “corresponding to,” asthey relate to sequence alignment, are intended to mean enumeratedpositions within a reference protein, e.g., p70S6K (SEQ ID NO:7), andthose positions that align with the positions on the reference protein.Thus, when the amino acid sequence of a subject peptide is aligned withthe amino acid sequence of a reference peptide, e.g., SEQ ID NO:7, theamino acids in the subject peptide sequence that “correspond to” certainenumerated positions of the reference peptide sequence are those thatalign with these positions of the reference peptide sequence, but arenot necessarily in these exact numerical positions of the referencesequence. Methods for aligning sequences for determining correspondingamino acids between sequences are described below.

Additional embodiments of the invention include monomers (as describedabove) based on any putative or real substrate for mTOR, such assubstrates identified by SEQ ID NOS:7-13. Furthermore, if the substratehas more than one recognition motif, then more than one monomer may beidentified therein.

Another embodiment of the invention is a nucleic acid moleculecomprising a polynucleotide sequence encoding at least one copy of aligand peptide.

Another embodiment of the invention is an isolated polypeptidehomopolyligand, wherein the homopolyligand modulates mTOR activity.

Another embodiment of the invention is an isolated polypeptideheteropolyligand, wherein the heteropolyligand modulates mTOR activity.

Another embodiment of the invention is a nucleic acid molecule whereinthe polynucleotide sequence encodes one or more copies of one or morepeptide ligands.

Another embodiment of the invention is a nucleic acid molecule whereinthe polynucleotide sequence encodes at least a number of copies of thepeptide selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or10.

Another embodiment of the invention is a vector comprising a nucleicacid molecule encoding at least one copy of a ligand or polyligand.

Another embodiment of the invention is a recombinant host cellcomprising a vector comprising a nucleic acid molecule encoding at leastone copy of a ligand or polyligand.

Another embodiment of the invention is a method of inhibiting mTOR in acell comprising transfecting a vector comprising a nucleic acid moleculeencoding at least one copy of a ligand or polyligand into a host celland culturing the transfected host cell under conditions suitable toproduce at least one copy of the ligand or polyligand.

The invention also relates to modified inhibitors that are at leastabout 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identical to a referenceinhibitor. A “modified inhibitor” is used to mean a peptide that can becreated by addition, deletion or substitution of one or more amino acidsin the primary structure (amino acid sequence) of a inhibitor protein orpolypeptide. A “modified recognition motif” is a naturally occurringmTOR recognition motif that has been modified by addition, deletion, orsubstitution of one or more amino acids in the primary structure (aminoacid sequence) of the motif. For example, a modified mTOR recognitionmotif may be a motif where the phosphorylatable amino acid has beenmodified to a non-phosphorylatable amino acid. The terms “protein,”“peptide” and “polypeptide” are used interchangeably herein. Thereference inhibitor is not necessarily a wild-type protein or a portionthereof. Thus, the reference inhibitor may be a protein or peptide whosesequence was previously modified over a wild-type protein. The referenceinhibitor may or may not be the wild-type protein from a particularorganism.

A polypeptide having an amino acid sequence at least, for example, about95% “identical” to a reference an amino acid sequence is understood tomean that the amino acid sequence of the polypeptide is identical to thereference sequence except that the amino acid sequence may include up toabout five modifications per each 100 amino acids of the reference aminoacid sequence encoding the reference peptide. In other words, to obtaina peptide having an amino acid sequence at least about 95% identical toa reference amino acid sequence, up to about 5% of the amino acidresidues of the reference sequence may be deleted or substituted withanother amino acid or a number of amino acids up to about 5% of thetotal amino acids in the reference sequence may be inserted into thereference sequence. These modifications of the reference sequence mayoccur at the N-terminus or C-terminus positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among amino acids in the reference sequence or inone or more contiguous groups within the reference sequence.

As used herein, “identity” is a measure of the identity of nucleotidesequences or amino acid sequences compared to a reference nucleotide oramino acid sequence. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per sc has an art-recognizedmeaning and can be calculated using published techniques. (See, e.g.,Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics And Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); von Heinje, G., Sequence Analysis In MolecularBiology, Academic Press (1987); and Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M Stockton Press, New York (1991)). Whilethere exist several methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math48:1073 (1988)). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994) and Carillo, H. & Lipton, D., Siam J AppliedMath 48:1073 (1988). Computer programs may also contain methods andalgorithms that calculate identity and similarity. Examples of computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP,ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403(1990)) and FASTDB. Examples of methods to determine identity andsimilarity are discussed in Michaels, G. and Garian, R., CurrentProtocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000),which is incorporated by reference. In one embodiment of the presentinvention, the algorithm used to determine identity between two or morepolypeptides is BLASTP.

In another embodiment of the present invention, the algorithm used todetermine identity between two or more polypeptides is FASTDB, which isbased upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990), incorporated by reference). In a FASTDB sequence alignment, thequery and subject sequences are amino sequences. The result of sequencealignment is in percent identity. Parameters that may be used in aFASTDB alignment of amino acid sequences to calculate percent identityinclude, but are not limited to: Matrix=PAM, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject amino sequence, whichever is shorter.

If the subject sequence is shorter or longer than the query sequencebecause of N-terminus or C-terminus additions or deletions, not becauseof internal additions or deletions, a manual correction can be made,because the FASTDB program does not account for N-terminus andC-terminus truncations or additions of the subject sequence whencalculating percent identity. For subject sequences truncated at bothends, relative to the query sequence, the percent identity is correctedby calculating the number of amino acids of the query sequence that areN-and C-terminus to the reference sequence that are not matched/aligned,as a percent of the total amino acids of the query sequence. The resultsof the FASTDB sequence alignment determine matching/alignment. Thealignment percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score can beused for the purposes of determining how alignments “correspond” to eachother, as well as percentage identity. Residues of the query (subject)sequences or the reference sequence that extend past the N— or C-terminiof the reference or subject sequence, respectively, may be consideredfor the purposes of manually adjusting the percent identity score. Thatis, residues that are not matched/aligned with the N— or C-termini ofthe comparison sequence may be counted when manually adjusting thepercent identity score or alignment numbering.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue reference sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a match/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N— and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 reference sequence. This time thedeletions are internal deletions so there are no residues at the N— orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected.

The polyligands of the invention optionally comprise spacer amino acidsbefore, after, or between monomers. The length and composition of thespacer may vary. An example of a spacer is glycine, alanine,polyglycine, or polyalanine. Specific examples of spacers used betweenmonomers in SEQ ID NO:1 are the four amino acid spacers PAAA and PAGA,and the six amino acid spacer EFPGGG. In the instance of SEQ ID NO:1,the proline-containing spacer is intended to break an alpha helicalsecondary structure. Spacer amino acids may be any amino acid and arenot limited to these alanine, glycine, and proline-containing examples.The instant invention is directed to all combinations of homopolyligandsand heteropolyligands, with or without spacers, and without limitationto the examples given above or below.

The ligands and polyligands of the invention are optionally linked toadditional molecules or amino acids that provide an epitope tag, areporter, and/or localize the ligand to a region of a cell (See FIGS.5A-5G, FIGS. 6A-6G, FIGS. 7A-7G, and FIGS. 8A-8G). Non-limiting examplesof epitope tags are FLAG™ (Kodak; Rochester, N.Y.), HA (hemagluttinin),c-Myc and His6. Non-limiting examples of reporters are alkalinephosphatase, galactosidase, peroxidase, luciferase and green fluorescentprotein (GFP). Non-limiting examples of cellular localizations aresarcoplamic reticulum, endoplasmic reticulum, mitochondria, golgiapparatus, nucleus, plasma membrane, apical membrane, and basolateralmembrane. The epitopes, reporters and localization signals are given byway of example and without limitation. The epitope tag, reporter and/orlocalization signal may be the same molecule. The epitope tag, reporterand/or localization signal may also be different molecules.

Ligands and polyligands and optional amino acids linked thereto can besynthesized chemically or recombinantly using techniques known in theart. Chemical synthesis techniques include but are not limited topeptide synthesis which is often performed using an automated peptidesynthesizer. Pepetides can also be synthesized utilizing non-automatedpeptide sythesis methods known in the art. Recombinant techniquesinclude insertion of ligand-encoding nucleic acids into expressionvectors, wherein nucleic acid expression products are synthesized usingcellular factors and processes.

Linkage of a cellular localization signal, epitope tag, or reporter to aligand or polyligand can include covalent or enzymatic linkage to theligand. When the localization signal comprises material other than apolypeptide, such as a lipid or carbohydrate, a chemical reaction tolink molecules may be utilized. Additionally, non-standard amino acidsand amino acids modified with lipids, carbohydrates, phosphate or othermolecules may be used as precursors to peptide synthesis. The ligands ofthe invention have therapeutic utility with or without localizationsignals. However, ligands linked to localization signals have utility assubcellular tools or therapeutics. For example, ligands depictedgenerically in FIGS. 7A-7G represent ligands with utility as subcellulartools or therapeutics. mTOR ligand-containing gene constructs are alsodelivered via gene therapy. FIGS. 10B and 10C depict embodiments of genetherapy vectors for delivering and controlling polypeptide expression invivo. Polynucleotide sequences linked to the gene construct in FIGS. 10Band 10C include genome integration domains to facilitate integration ofthe transgene into a viral genome and/or host genome.

FIG. 10A shows a vector containing an mTOR ligand gene construct,wherein the ligand gene construct is releasable from the vector as aunit useful for generating transgenic animals. For example, the ligandgene construct, or transgene, is released from the vector backbone byrestriction endonuclease digestion. The released transgene is theninjected into pronuclei of fertilized mouse eggs; or the transgene isused to transform embryonic stem cells. The vector containing a ligandgene construct of FIG. 10A is also useful for transient transfection ofthe trangene, wherein the promoter and codons of the transgene areoptimized for the host organism. The vector containing a ligand geneconstruct of FIG. 10A is also useful for recombinant expression ofpolypeptides in fermentable organisms adaptable for small or large scaleproduction, wherein the promoter and codons of the transgene areoptimized for the fermentation host organism.

FIG. 10D shows a vector containing an mTOR ligand gene construct usefulfor generating stable cell lines.

The invention also encompasses polynucleotides comprising nucleotidesequences encoding ligands, homopolyligands, and heteropolyligands. Thepolynucleotides of the invention are optionally linked to additionalnucleotide sequences encoding epitopes, reporters and/or localizationsignals. Further, the nucleic acids of the invention are optionallyincorporated into vector polynucleotides. The polynucleotides areoptionally flanked by nucleotide sequences comprising restrictionendonuclease sites and other nucleotides needed for restrictionendonuclese activity. The flanking sequences optionally provide cloningsites within a vector. The restriction sites can include, but are notlimited to, any of the commonly used sites in most commerciallyavailable cloning vectors. Sites for cleavage by other restrictionenzymes, including homing endonucleases, are also used for this purpose.The polynucleotide flanking sequences also optionally providedirectionality of subsequence cloning. It is preferred that 5′ and 3′restriction endonuclease sites differ from each other so thatdouble-stranded DNA can be directionally cloned into correspondingcomplementary sites of a cloning vector.

Ligands and polyligands with or without localization signals, epitopesor reporters are alternatively synthesized by recombinant techniques.Polynucleotide expression constructs are made containing desiredcomponents and inserted into an expression vector. The expression vectoris then transfected into cells and the polypeptide products areexpressed and isolated. Ligands made according to recombinant DNAtechniques have utility as research tools and/or therapeutics.

The following is an example of how polynucleotides encoding ligands andpolyligands are produced. Complimentary oligonucleotides encoding theligands and flanking sequences are synthesized and annealled. Theresulting double-stranded DNA molecule is inserted into a cloning vectorusing techniques known in the art. When the ligands and polyligands areplaced in-frame adjacent to sequences within a transgenic gene constructthat is translated into a protein product, they form part of a fusionprotein when expressed in cells or transgenic animals.

Another embodiment of the invention relates to selective control oftransgene expression in a desired cell or organism. The promotor portionof the recombinant gene can be a constitutive promotor, anon-constitutive promotor, a tissue-specific promotor (constitutive ornon-constitutive) or a selectively controlled promotor. Differentselectively controlled promoters are controlled by different mechanisms.For example, RheoSwitch® is an inducible promotor system available fromRheoGene. Temperature sensitive promotors can also be used to increaseor decrease gene expression. An embodiment of the invention comprises aligand or polyligand gene construct whose expression is controlled by aninducible promotor. In one embodiment, the inducible promotor istetracycline controllable.

Polyligands are modular in nature. An aspect of the instant invention isthe combinatorial modularity of the disclosed polyligands. Anotheraspect of the invention are methods of making these modular polyligandseasily and conveniently. In this regard, an embodiment of the inventioncomprises methods of modular subsequence cloning of genetic expressioncomponents. When the ligands, homopolyligands, heteropolyligands andoptional amino acid expression components are synthesized recombinantly,one can consider each clonable element as a module. For speed andconvenience of cloning, it is desirable to make modular elements thatare compatible at cohesive ends and are easy to insert and clonesequentially. This is accomplished by exploiting the natural propertiesof restriction endonuclease site recognition and cleavage. One aspect ofthe invention encompasses module flanking sequences that, at one end ofthe module, are utilized for restriction enzyme digestion once, and atthe other end, utilized for restriction enzyme digestion as many timesas desired. In other words, a restriction site at one end of the moduleis utilized and destroyed in order to effect sequential cloning ofmodular elements. An example of restriction sites flanking a codingregion module are sequences recognized by the restriction enzymes NgoMIV and Cla I; or Xma I and Cla I. Cutting a first circular DNA with NgoMIV and Cla I to yield linear DNA with a 5′ NgoM IV overhang and a 3′ ClaI overhang; and cutting a second circular DNA with Xma I and Cla I toyield linear DNA with a 5′ Cla I overhang and a 3′ Xma I overhanggenerates first and second DNA fragments with compatible cohesive ends.When these first and second DNA fragments are mixed together, annealed,and ligated to form a third circular DNA fragment, the NgoM IV site thatwas in the first DNA and the Xma I site that was in the second DNA aredestroyed in the third circular DNA. Now this vestigial region of DNA isprotected from further Xma I or NgoM IV digestion, but flankingsequences remaining in the third circular DNA still contain intact 5′NgoM IV and 3′ Cla I sites. This process can be repeated numerous timesto achieve directional, sequential, modular cloning events. Restrictionsites recognized by NgoM IV, Xma I, and Cla I endonucleases represent agroup of sites that permit sequential cloning when used as flankingsequences.

Another way to assemble coding region modules directionally andsequentially employs linear DNA in addition to circular DNA. Forexample, like the sequential cloning process described above,restriction sites flanking a coding region module are sequencesrecognized by the restriction enzymes NgoM IV and Cla I; or Xma I andCla I. A first circular DNA is cut with NgoM IV and Cla I to yieldlinear DNA with a 5′ NgoM IV overhang and a 3′ Cla I overhang. A secondlinear double-stranded DNA is generated by PCR amplification or bysynthesizing and annealing complimentary oligonucleotides. The secondlinear DNA has 5′ Cla I overhang and a 3′ Xma I overhang, which arecompatible cohesive ends with the first DNA linearized. When these firstand second DNA fragments are mixed together, annealed, and ligated toform a third circular DNA fragment, the NgoM IV site that was in thefirst DNA and the Xma I site that was in the second DNA are destroyed inthe third circular DNA. Flanking sequences remaining in the thirdcircular DNA still contain intact 5′ NgoM IV and 3′ Cla I sites. Thisprocess can be repeated numerous times to achieve directional,sequential, modular cloning events. Restriction sites recognized by NgoMIV, Xma I, and Cla I endonucleases represent a group of sites thatpermit sequential cloning when used as flanking sequences. This processis depicted in FIG. 11.

One of ordinary skill in the art recognizes that other restriction sitegroups can accomplish sequential, directional cloning as describedherein. Preferred criteria for restriction endonuclease selection areselecting a pair of endonucleases that generate compatible cohesive endsbut whose sites are destroyed upon ligation with each other. Anothercriteria is to select a third endouclease site that does not generatesticky ends compatible with either of the first two. When such criteriaare utilized as a system for sequential, directional cloning, ligands,polyligands and other coding regions or expression components can becombinatorially assembled as desired. The same sequential process can beutilized for epitope, reporter, and/or localization signals.

Polyligands and methods of making polyligands that modulate mTORactivity are disclosed. Therapeutics include delivery of purified ligandor polyligand with or without a localization signal to a cell.Alternatively, ligands and polyligands with or without a localizationsignals are delivered via adenovirus, lentivirus, adeno-associatedvirus, or other viral constructs that express protein product in a cell.

Assays. Ligands of the invention are assayed for kinase modulatingactivity using one or more of the following exemplary methods.

Method 1. A biochemical assay is performed employingcommercially-obtained kinase, commercially-obtained substrate,commercially-obtained kinase inhibitor (control), and semi-purifiedinhibitor ligand of the invention (decoy ligand). Ligands (also referredto herein as decoy ligands) are linked to an epitope tag at one end ofthe polypeptide for purification and/or immobilzation, for example, on amicrotiter plate. The tagged decoy ligand is made using an in vitrotranscription/translation system such as a reticulocyte lysate systemwell known in the art. A vector polynucleotide comprising a promotor,such as T7 and/or T3 and/or SP6 promotor, a decoy ligand codingsequence, and an epitope tag coding sequence is employed to synthesizethe tagged decoy ligand in an in vitro transcription/translation system.In vitro transcription/translation protocols are disclosed in referencemanuals such as: Current Protocols in Molecular Biology (eds. Ausubel etal., Wiley, 2004 edition.) and Molecular Cloning: A Laboratory Manual(Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001, thirdedition). Immunoreagent-containing methods such as western blots,elisas, and immunoprecipitations are performed as described in: UsingAntibodies: A Laboratory Manual (Harlow and Lane Cold Spring HarborLaboratory Press, 1999).

For example, tagged decoy ligand synthesized using an in vitrotranscription/translation system is semi-purified and added to amicrotiter plate containing kinase enzyme and substrate immobilized byan anti-substrate specific antibody. Microtiter plates are rinsed tosubstantially remove non-immobilized components. Kinase activity is adirect measure of the phosphorylation of substrate by kinase employing aphospho-substrate specific secondary antibody conjugated to horseradishperoxidase (HRP) followed by the addition of3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The catalysisof TMB by HRP results in a blue color that changes to yellow uponaddition of phosphoric or sulfuric acid with a maximum absorbance at 450nm. The Control experiments include absence of kinase enzyme, and/orabsence of decoy ligand, and/or presence/absence of known kinaseinhibitors. A known kinase inhibitor useful in the assay isstaurosporine.

Method 2. A similar assay is performed employing the same reagents asabove but the substrate is biotinylated and immobilized by binding to astreptavidin-coated plate.

Method 3. A biochemical assay is performed employingcommercially-obtained kinase, commercially-obtained substrate,commercially-obtained kinase inhibitor (control), and semi-purifiedinhibitor ligand of the invention (decoy ligand) in a microtiter plate.A luminescent-based detection system, such as Promega's Kinase-Glo, isthen added to measure kinase activity.

For example, tagged decoy ligand synthesized using an in vitrotranscription/translation system is semi-purified and added to amicrotiter plate containing kinase enzyme and substrate. After thekinase assay is performed, luciferase and luciferin are added to thereaction. Luciferase utilizes any remaining ATP not used by the kinaseto catalyze luciferin. The luciferase reaction results in the productionof light which is related to kinase activity. Control experimentsinclude absence of kinase enzyme, and/or absence of decoy ligand, and/orpresence/absence of known kinase inhibitors. A known kinase inhibitoruseful in the assay is staurosporine.

Method 4. A similar cell-based assay is performed employing samereagents as above, but synthesizing the decoy ligand in a mammalian cellsystem instead of an in vitro transcription/translation system. Decoyligands are linked to an epitope tag at one end of the polypeptide forimmobilzation and/or for purification and/or for identification in awestern blot. Optionally, tagged decoy ligands are also linked to acellular localization signal for phenotypic comparison of pan-cellularand localized kinase modulation. A vector polynucleotide comprising aconstitutive promotor, such as the CMV promotor, a decoy ligand codingsequence, an epitope tag coding sequence, and optionally a localizationsignal coding sequence is employed to express the decoy ligand in cells.Transfection and expression protocols are disclosed in reference manualssuch as: Current Protocols in Molecular Biology (eds. Ausubel et al.,Wiley, 2004 edition.) and Molecular Cloning: A Laboratory Manual(Sambrook and Russell (Cold Spring Harbor Laboratory Press, 2001, thirdedition). Western Blots and immunoreagent-containing methods areperformed as described in: Using Antibodies: A Laboratory Manual (Harlowand Lane Cold Spring Harbor Laboratory Press, 1999).

EXAMPLES Example 1

A polypeptide comprising a heteropolyligand, an endoplasmic reticulumcellular localization signal, and a His6 epitope is synthesized.Examples of such polypeptides are generically represented by FIGS. 8A,8B, 8D, 8E and 8F. The polypeptide is synthesized on an automatedpeptide synthesizer or is recombinantly expressed and purified. Purifiedpolypeptide is solubilized in media and added to cells. The polypeptideis endocytosed by the cells, and transported to the endoplasmicreticulum. Verification is performed by immunohistochemical stainingusing an anti-His6 antibody.

Example 2

A transgene is constructed using a cytomegalovirus (CMV) promoter todirect expression of a fusion protein comprising SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:37, wherein Xaa is alanine (POLYLIGAND), greenfluorescent protein (REPORTER), and a plasma membrane localizationsignal (LOCALIZATION SIGNAL). Such a transgene is genericallyrepresented by FIG. 9C. The transgene is transfected into cells fortransient expression. Verification of expression and location isperformed by visualization of green fluorescent protein by confocalmicroscopy.

Example 3

A transgene construct is built to produce a protein product withexpression driven by a tissue-specific promoter. The transgene comprisesa synthetic gene expression unit engineered to encode three domains.Each of these three domains is synthesized as a pair of complimentarypolynucleotides that are annealed in solution, ligated and inserted intoa vector. Starting at the amino-terminus, the three domains in theexpression unit are nucleotide sequences that encode an mTOR ligand, aFLAG™ epitope, and a nuclear localization signal. The mTOR ligand is amonomeric ligand, homopolymeric ligand or heteropolymeric ligand asdescribed herein. Nucleotide sequences encoding a FLAG™ epitope areplaced downstream of nucleotide sequences encoding the mTOR ligand.Finally, nucleotide sequences encoding the localization signal areplaced downstream of those encoding the FLAG™ epitope. The assembledgene expression unit is subsequently subcloned into an expressionvector, such as that shown in FIG. 10A, and used to transientlytransfect cells. Verification is performed by immunohistochemicalstaining using an anti-FLAG™ antibody.

Example 4

Modulation of mTOR cellular function by subcellularly localized mTORpolyligand is illustrated. A transgene construct containing nucleicacids that encode a polyligand fusion protein, epitope, and endoplasmicreticulum localization signal is made. The expression unit containsnucleotides that encode SEQ ID NO:1 (POLYLIGAND), a c-Myc epitope(EPITOPE), and a nuclear localization signal (LOCALIZATION SIGNAL). Thisexpression unit is subsequently subcloned into a vector between aEF1alpha promoter and an SV40 polyadenylation signal. The completedtransgene-containing expression vector is then used to transfect cells.Inhibition of mTOR activity is demonstrated by measuring phosphorylationof endogenous substrates against controls and/or observing phenotypes.

Example 5

Ligand function and localization is demonstrated in vivo by making atransgene construct used to generate mice expressing a ligand fusionprotein targeted to the nucleus. The transgene construct is showngenerically in FIG. 10B. The expression unit contains nucleotides thatencode a tetramer of SEQ ID NO:33, a hemagluttinin epitope, and anuclear localization signal. This expression unit is subsequentlysubcloned into a vector between nucleotide sequences including aninducible promoter and an SV40 polyadenylation signal. The completedtransgene is then injected into pronuclei of fertilized mouse oocytes.The resultant pups are screened for the presence of the transgene byPCR. Transgenic founder mice are bred with wild-type mice. Heterozygoustransgenic animals from at least the third generation are used for thefollowing tests, with their non-transgenic littermates serving ascontrols.

Test 1: Southern blotting analysis is performed to determine the copynumber. Southern blots are hybridized with a radio-labeled probegenerated from a fragment of the transgene. The probe detects bandscontaining DNA from transgenic mice, but does not detect bandscontaining DNA from non-transgenic mice. Intensities of the transgenicmice bands are measured and compared with the transgene plasmid controlbands to estimate copy number. This demonstrates that mice in Example 5harbor the transgene in their genomes.

Test 2: Tissue homogenates are prepared for Western blot analysis. Thisexperiment demonstrates the transgene is expressed in tissues oftransgenic mice because hemagluttinin epitope is detected in transgenichomogenates but not in non-transgenic homogenates.

Test 3: Function is assessed by phenotypic observation or analysisagainst controls after induction of expression.

These examples demonstrate delivery of ligands to a localized region ofa cell for therapeutic or experimental purposes. The purifiedpolypeptide ligands can be formulated for oral or parenteraladministration, topical administration, or in tablet, capsule, or liquidform, intranasal or inhaled aerosol, subcutaneous, intramuscular,intraperitoneal, or other injection; intravenous instillation; or anyother routes of administration. Furthermore, the nucleotide sequencesencoding the ligands permit incorporation into a vector designed todeliver and express a gene product in a cell. Such vectors includeplasmids, cosmids, artificial chromosomes, and modified viruses.Delivery to eukaryotic cells can be accomplished in vivo or ex vivo. Exvivo delivery methods include isolation of the intended recipient'scells or donor cells and delivery of the vector to those cells, followedby treatment of the recipient with the cells.

Disclosed are ligands and polyligands that modulate mTOR activity andmethods of making and using these ligands. The ligands and polyligandsare synthesized chemically or recombinantly and are utilized as researchtools or as therapeutics. The invention includes linking the ligands andpolyligands to cellular localization signals for subcellulartherapeutics.

1. An isolated polypeptide heteropolyligand comprising an amino acidsequence at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 4, whereinthe heteropolyligand modulates mTOR activity.
 2. The isolatedpolypeptide heteropolyligand of claim 1, wherein said heteropolyligandcomprises two or more monomeric polypeptide ligands comprising an aminoacid sequence selected from any one of SEQ ID NOS: 14, 15, 16, 22, 23,24, 31 and 54, wherein Xaa is any amino acid.
 3. An isolated fusionpolypeptide comprising two or more polypeptides selected from SEQ IDNOS: 14, 15, 16, 22, 23, 24, 31 and 54, wherein Xaa is any amino acid,wherein the fusion polypeptide modulates mTOR activity.
 4. The isolatedfusion polypeptide of claim 3, wherein at least one amino aciddesignated as Xaa is an amino acid other than serine or threonine. 5.The isolated fusion polypeptide of claim 3, wherein at least one aminoacid designated as Xaa is alanine.
 6. The isolated polypeptideheteropolyligand of claim 2, wherein said heteropolyligand comprises theamino acid sequences of SEQ ID NOS: 22, 24, 31 and
 54. 7. The isolatedpolypeptide heteropolyligand of claim 2, wherein said heteropolyligandcomprises the amino acid sequences of SEQ ID NOS: 14, 15, 16 and
 23. 8.The isolated fusion polypeptide of claim 3, wherein saidheteropolyligand comprises the amino acid sequences of SEQ ID NOS: 22,24, 31 and
 54. 9. The isolated fusion polypeptide of claim 3, whereinsaid heteropolyligand comprises the amino acid sequences of SEQ ID NOS:14, 15, 16 and
 23. 10. The isolated polypeptide heteropolyligand ofclaim 2, wherein at least one amino acid designated as Xaa is an aminoacid other than serine or threonine.
 11. The isolated polypeptideheteropolyligand of claim 6, wherein at least one amino acid designatedas Xaa is an amino acid other than serine or threonine.
 12. The isolatedpolypeptide heteropolyligand of claim 7, wherein at least one amino aciddesignated as Xaa is an amino acid other than serine or threonine. 13.The isolated fusion polypeptide of claim 8, wherein at least one aminoacid designated as Xaa is an amino acid other than serine or threonine.14. The isolated fusion polypeptide of claim 9, wherein at least oneamino acid designated as Xaa is an amino acid other than serine orthreonine.
 15. The isolated polypeptide heteropolyligand of claim 2,wherein at least one amino acid designated as Xaa is alanine.
 16. Theisolated polypeptide heteropolyligand of claim 6, wherein at least oneamino acid designated as Xaa is alanine.
 17. The isolated polypeptideheteropolyligand of claim 7, wherein at least one amino acid designatedas Xaa is alanine.
 18. The isolated fusion polypeptide of claim 8,wherein at least one amino acid designated as Xaa is alanine.
 19. Theisolated fusion polypeptide of claim 9, wherein at least one amino aciddesignated as Xaa is alanine.